Name: 作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略
Uploaded: 2024-03-01T14:15:00
Description: Watch this Scientific Journal Video about Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model at JoVE.com

我们要感谢斯坦福大学Wu Tsai神经科学显微镜服务中心，斯坦福大学体内成像创新中心（SCi3） - 小动物成像中心，NIH S10共享仪器资助（S10RR026917-01，PI Michael Moseley博士）和斯坦福大学临床前成像设施为该项目提供设备和基础设施。这项工作得到了国家儿童健康与人类发展研究所的资助，资助编号为R01HD103638。我们要感谢斯坦福大学的Schnitzer集团;圣克鲁斯大学左实验室;以及波士顿大学波士顿光子中心神经血管成像实验室，就双光子成像和颅窗模型进行教育讨论。

本协议中描述的动物程序符合实验动物护理管理小组 （APLAC） 的要求。
1. 细胞培养
<ol><li>罩子的准备<ol><li>洗手，戴手套和实验室外套。打开生物安全柜并将窗扇水平设置为适当的打开高度。让抽油烟机吹扫 3-5 分钟。用 70% 乙醇喷洒引擎盖区域，然后用纸巾擦拭。</li>
		<li>用70%乙醇喷洒所有试剂，并用薄纸擦拭。将试剂移入罩中。不要在烤架上放任何物品。如...

在小鼠中使用 2P-IVM 实时跟踪脑肿瘤中的纳米颗粒

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+of+Paneth+cells+in+the+small+intestine+of+live+mice.">Two-photon microscopy of Paneth cells in the small intestine of live mice.</a> Sci Rep. 8 (1), 14174 (2018).</li><li>Ihler, F., Bertlich, M., Weiss, B., Dietzel, S., Canis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+allows+imaging+and+characterization+of+cochlear+microvasculature+in+vivo.">Two-photon microscopy allows imaging and characterization of cochlear microvasculature in vivo.</a> Biomed Res Int. 2015, 154272 (2015).</li><li>Matsuura, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+with+two-photon+microscopy+reveals+cellular+dynamics+in+the+ischeamia-reperfused+rat+heart.">Intravital imaging with two-photon microscopy reveals cellular dynamics in the ischeamia-reperfused rat heart.</a> Sci Rep. 8 (1), 15991 (2018).</li><li>Veres, T. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intubation-free+in+vivo+imaging+of+the+tracheal+mucosa+using+two-photon+microscopy.">Intubation-free in vivo imaging of the tracheal mucosa using two-photon microscopy.</a> Sci Rep. 7 (1), 694 (2017).</li><li>Zong, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+two-photon+calcium+imaging+in+freely+moving+mice.">Large-scale two-photon calcium imaging in freely moving mice.</a> Cell. 185 (7), 1240-1256.e30 (2022).</li><li>Takasaki, K., Abbasi-Asl, R., Waters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superficial+bound+of+the+depth+limit+of+two-photon+imaging+in+mouse+brain.">Superficial bound of the depth limit of two-photon imaging in mouse brain.</a> eNeuro. 17 (1), (2019).</li><li>Birkner, A., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+two-photon+imaging+in+vivo+with+a+red-shifted+calcium+indicator.">Deep two-photon imaging in vivo with a red-shifted calcium indicator.</a> Methods Mol Biol. 1929, 15-26 (1929).</li><li>Busche, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+imaging:+live+imaging+of+glioma+by+two-photon+microscopy.">Brain tumor imaging: live imaging of glioma by two-photon microscopy.</a> Cold Spring Harb Protoc. 2013 (3), (2013).</li><li>Ricard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+dynamics+of+microglial+and+monocyte-derived+cells+in+glioblastoma-bearing+mice.">Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice.</a> Sci Rep. 6, 26381 (2016).</li><li>Soub&#233;ran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+VEGF+blockade+on+the+dynamics+of+the+inflammatory+landscape+in+glioblastoma-bearing+mice.">Effects of VEGF blockade on the dynamics of the inflammatory landscape in glioblastoma-bearing mice.</a> J Neuroinflammation. 16 (1), 191 (2019).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+vivo+reveals+specific+nanoparticle+target+binding+in+a+syngeneic+glioma+mouse+model.">Real-time vivo reveals specific nanoparticle target binding in a syngeneic glioma mouse model.</a> Nanoscale. 14 (15), 5678-5688 (2022).</li><li>Wartchow, K. 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L., Hambardzumyan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+2-photon+imaging+reveals+distinct+morphology+and+infiltrative+properties+of+glioblastoma-associated+macrophages.">Intravital 2-photon imaging reveals distinct morphology and infiltrative properties of glioblastoma-associated macrophages.</a> Proc Natl Acad Sci U S A. 116 (28), 14254-14259 (2019).</li><li>Zhang, Y. 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我们提出了一种使用 2P-IVM 通过颅窗进行实时 体内 NP 跟踪的方法，以评估荧光标记的氧化铁 NP 的肿瘤递送。该手术的手术技术需要稳定的手和先进的实验手术技能。建议在继续体验活体动物之前练习使用尸体或幻影。作为替代方案，Hoeferlin等人实施了机器人钻头，以减少热损伤，最大限度地减少手术技术的可变性，并使手术标准化22。
窗口的大小表?...

双光子活体显微镜 （2P-IVM） 是一种荧光成像技术，可实现活组织1 的可视化。
2P-IVM 于 1990 年代首次开发，已用于视网膜2、肾脏3、小肠4、耳蜗5、心脏6、气管7 和各种临床前模型的大脑的体内分析 8,9。在神经科学领域，2P-IVM 作为一种对清醒动物健康大脑进行实时成像的技术10 以及研究神经系统疾病（如阿尔茨海默氏症11、帕金森氏症12 和胶质母细胞瘤 （GBM）13、14、15、16）的重要性。
2P-IVM为研究GBM发展过程中的肿瘤微环境提供了一种优雅的解决方案。虽然以前的一些研究集中在 体外17 和 离体 模型18，但其他研究则实施了原位19 和异源性20 体内 模型来检查 GBM。Madden 等人在小鼠模型13 中对 CNS-1 大鼠神经胶质瘤细胞系进行了天然成像。Ricard等人使用原位GL261-DsRed小鼠模型，静脉注射荧光基团以增强2P-IVM14中肿瘤区域的血管。
在这里，我们应用 2P-IVM 来跟踪 GBM 原位小鼠模型中荧光标记的氧化铁纳米颗粒 （NP） 的肿瘤递送。使用颅窗，这种方法使我们能够详细研究大脑中NP的实时时空分布。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium chloride infusion solution</td>
			<td>Baxter Corp</td>
			<td>533-JB1301P</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringes</td>
			<td>BD</td>
			<td>Luer-Lok syringe, REF309628</td>
			<td></td>
		</tr>
		<tr>
			<td>10%&#160;FBS</td>
			<td>Thermo fisher</td>
			<td>Cytiva SH30910.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>10% DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>2-photon microscope</td>
			<td>Prairie Technologies, Bruker</td>
			<td>Prairie Ultima IV</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol applicators, 70%</td>
			<td>Medline Industries, LP</td>
			<td>MDS093810</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol, spray bottle</td>
			<td>Decon Labs Inc</td>
			<td>Decon SaniHol, 04-355-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynold Brands</td>
			<td>Reynold Wrap non-stick aluminum foil</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia machine</td>
			<td>Patterson Scientific</td>
			<td>SAS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia monitoring&#160;</td>
			<td>Kent Scientific&#160;</td>
			<td>MouseSTAT Jr. Rodent Pulsoxymeter</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x), liquid</td>
			<td>Invitrogen</td>
			<td>15240-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine applicators</td>
			<td>Professional Disposables International, Inc</td>
			<td>S41125</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch, 5 mm&#160;</td>
			<td>Miltex</td>
			<td>Size 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine sustained release</td>
			<td>Zoo Pharm</td>
			<td>Bup SR Lab, 1.0 mg/mL</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>C6 rat glioma cell line</td>
			<td>ATCC (American Tissue Culture Collection)</td>
			<td>CCL-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannulas</td>
			<td>BD</td>
			<td>16 G, 1.1/2&#8221;, 30 G, 1&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer&#160;</td>
			<td>Rimadyl, 50 mg/ml</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>Cefazoline</td>
			<td>Sagent Pharmaceuticals</td>
			<td>25021-101-10, 1 g/vial</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>87711</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tip applicators, 6&#8221;&#160;</td>
			<td>Dyad Medical Sourcing, LLC</td>
			<td>HCS1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Stoelting Co</td>
			<td>51459</td>
			<td>Dental cement kit, clear, 2 components</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Bimeda</td>
			<td>138RX, 2 mg/mL</td>
			<td>A generic drug can be used instead.&#160;</td>
		</tr>
		<tr>
			<td>DietGel</td>
			<td>ClearH2O</td>
			<td>Recovery, 72-06-502</td>
			<td></td>
		</tr>
		<tr>
			<td>Drape&#160;</td>
			<td>Cardinal Health</td>
			<td>Bio Shield Wrap</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Saeyang Microtech</td>
			<td>Escort Pro, B08350</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill tips&#160;</td>
			<td>Hager &#38; Meissinger GmbH</td>
			<td>REF310104001001005</td>
			<td>Size 005, US 1/4</td>
		</tr>
		<tr>
			<td>FIJI imaging analysis software</td>
			<td>National Institute of Health</td>
			<td>https://imagej.net/software/fiji/</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>13-812-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisher HealthCare</td>
			<td>Sterile Cotton Gauze Pad, 4 x 4&#8221;, 22-415-469</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam&#160;</td>
			<td>Ethicon Inc.&#160;</td>
			<td>Surgifoam absorbable gelatin sponge, Ref. 1972</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator&#160;</td>
			<td>Cellpoint Scientific&#160;</td>
			<td>Germinator 500, No. 11688</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips, 5 mm diameter</td>
			<td>Fisher Scientific</td>
			<td>Menzel Cover glass&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves, non-sterile</td>
			<td>Fisher Scientific</td>
			<td>Nitrile powder-free medical examination gloves</td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves, sterile</td>
			<td>Medline Industries, LP</td>
			<td>MDS104070</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Church &#38; Dwight</td>
			<td>Nair Hair remover lotion&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton Company Inc</td>
			<td>Gastight #1701, 10 &#181;L</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS without Ca, Mg</td>
			<td>Fisher Scientific</td>
			<td>PI88284</td>
			<td></td>
		</tr>
		<tr>
			<td>Head bar</td>
			<td>Hongway</td>
			<td>5 mm inner diameter O-rings</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting Co.&#160;</td>
			<td>Rodent warmer X2</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>Exel International Medical Products&#160;</td>
			<td>29G x 1/2&#8243;</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron oxide nanoparticles</td>
			<td>Covis Pharma GmbH</td>
			<td>Feraheme ferumoxytol injection, 510 mg/17 mL, 59338077501</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Dechra</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Jackson Laboratories</td>
			<td>NSG, Strain 005557</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (surgery)</td>
			<td>Seiler Medical</td>
			<td>Seiler IQ Q-100-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticles</td>
			<td>Custom</td>
			<td></td>
			<td>Iron oxide nanoparticles (Ferumoxytol) labeled with fluorescein isothiocyanate</td>
		</tr>
		<tr>
			<td>Ophtalmic ointment</td>
			<td>Major pharmaceuticals</td>
			<td>Lubrifresh P.M. nighttime ointment, 203964</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>Linde Gas &#38; Equipment Inc.&#160;</td>
			<td>High Pressure Steel K Style Cylinder, 249CF, 2000PSIG, CGA 540</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic cups</td>
			<td>Georgia-Pacirif Consumer Products</td>
			<td>Dixie Portion Cup, 2 oz., Plastic, Clear, PK2400</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Braintree Scientific</td>
			<td>50-195-5494</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Ohaus Corp</td>
			<td>CR2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Integra Life Sciences Production Corp</td>
			<td>Integra Miltex Stainless steel disposable scalpel</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fisher Scientific</td>
			<td>13-804-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealant</td>
			<td>Henkel Corp</td>
			<td>Loctite 4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Single use lab gown</td>
			<td>High Tech Conversions</td>
			<td>17-444-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Stoelting Co.&#160;</td>
			<td>Stoelting New Standard TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Vacuum Bottle Top Filtration Systems</td>
			<td>Fisher Scientific</td>
			<td>S2GPU05RE, MilliporeSigma NO.:S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam box</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Cardinal Health</td>
			<td>19-163-108</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical glue, 3M Vetbond tissues adhesive</td>
			<td>3M Animal Care Prodcuts</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein cathether</td>
			<td>Custom</td>
			<td></td>
			<td>Consists of two 30 G cannulas connected with sillicone tubing&#160;</td>
		</tr>
		<tr>
			<td>TrypLE Express (1x), no phenol red</td>
			<td>Invitrogen</td>
			<td>12604-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolett torch</td>
			<td>Spring sunshine technology</td>
			<td>Consciot</td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon intravital microscopy of glioblastoma in a murine model

静脉注射癌症治疗药物对脑肿瘤的递送受到血脑屏障的限制。一种在 体内 直接成像脑肿瘤中大分子积累和分布的方法将大大提高我们在临床前模型中理解和优化药物递送的能力。该协议描述了一种在胶质母细胞瘤（GBM）小鼠模型中使用双光子活体显微镜（2P-IVM）对静脉内施用的荧光标记纳米颗粒进行实时 体内 跟踪的方法。
该协议包含对程序的多步骤描述，包括实验动物的麻醉和镇痛、创建颅窗、GBM 细胞植入、放置头杆、进行 2P-IVM 研究以及长期随访研究的术后护理。我们展示了具有代表性的2P-IVM成像会议和图像分析，研究了该技术的优缺点，并讨论了潜在的应用。
该方法可以很容易地修改和适应 体内 临床前脑成像领域的不同研究问题。

Two-photon intravital microscopy (2P-IVM) is a fluorescence imaging technique that allows the visualization of living tissue1.
First developed in the 1990s, 2P-IVM has been used for in vivo analysis of the retina2, kidney3, small intestine4, cochlea5, heart6, trachea7, and the brain in various preclinical models8,9. In the field of neuroscience, 2P-IVM has gained importance as a technique for real-time imaging of the healthy brain in awake animals10, as well as studying diseases of the nervous system such as Alzheimer&#39;s11, Parkinson&#39;s12 and glioblastoma (GBM)13,14,15,16.
2P-IVM offers an elegant solution for studying the tumor microenvironment during the development of GBM. While some previous studies focused on in vitro17 and ex vivo models18, others implemented orthotopic19 and xenotropic20&#160;in vivo models for examining GBM. Madden et al. performed native imaging of CNS-1 rat glioma cell line in a mouse model13. Using an orthotopic GL261-DsRed murine model, Ricard et al. performed an intravenous administration of a fluorophore to enhance the blood vessels in the tumor region in 2P-IVM14.
Here, we apply 2P-IVM for tracking the tumor delivery of fluorescent-labeled iron oxide nanoparticles (NP) in an orthotopic mouse model of GBM. Using a cranial window, this method allows us to study the real-time spatiotemporal distribution of NPs in the brain in detail.

作者聚焦：优化胶质母细胞瘤治疗中药物递送和早期检测的多模式成像策略

小鼠模型中胶质母细胞瘤的双光子活体显微镜

Glioblastoma is one of the most common primary brain cancer and is associated with fast growth and poor life expectancy. Developing effective therapies for glioblastoma remains a major challenge since the delivery of therapeutics is limited by the blood brain barrier. A method to directly image the accumulation and distribution of macromolecules in the brain would greatly enhance our ability to understand and optimize drug delivery.Two-photon microscopy offers an elegant solution for studying the real-time distribution of nanoparticles in the preclinical model of glioblastoma. By modifying the nanoparticle used in this study, it's possible to investigate the delivery of different drug candidates and combination therapies on a cellular and molecular level. In addition to two-photon intravital microscopy, the iron oxide component of these nanoparticles allow for multimodal imaging with MRI or MPI.For the future, imaging facilities could serve in the realization of early detection to cancer, thereby leading to more effective therapies against malignant neoplasia. One path to achieve this is to merge two-photon intravital microscopy with other imaging modalities such as positron emission tomography and photoacoustic ultrasound.

在这里，我们进行了颅窗手术，并将C6细胞移植到GBM的NSG小鼠模型中（n = 5）。在创建窗口（图1A）的所有组件之间建立适当的密封将确保窗口的长期成像的耐用性，并进一步降低发病率。使用适用于 体内 2P-IVM的载物台（图2），我们可以在麻醉下对动物进行长达2小时的成像，而不会出现任何重大运动伪影。在携带GBM的小鼠中静脉内施用纳米颗粒...

Watch this Scientific Journal Video about Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model at JoVE.com

我们提出了一种在小鼠模型中将荧光标记的氧化铁纳米颗粒递送至胶质母细胞瘤的双光子显微镜的新方法。

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

胶质母细胞瘤是最常见的原发性脑癌之一，与快速生长和预期寿命短有关。开发有效的胶质母细胞瘤疗法仍然是一个重大挑战，因为治疗药物的递送受到血脑屏障的限制。一种直接对大脑中大分子的积累和分布进行成像的方法将大大提高我们理解和优化药物递送的能力。双光子显微镜为研究胶质母细胞瘤临床前模型中纳米颗粒的实时分布提供了一种优雅的解决方案。通过修改本研究中使用的纳米颗粒，可以在细胞和分子水平上研究不同候选药物和联合疗法的递送。除了双光子活体显微镜外，这些纳米颗粒的氧化铁成分还允许使用MRI或MPI进行多模态成像。未来，成像设施可用于实现癌症的早期检测，从而为恶性肿瘤提供更有效的治疗方法。实现这一目标的一种途径是将双光子活体显微镜与其他成像方式（如正电子发射断层扫描和光声超声）合并。

two-photon-intravital-microscopy-of-glioblastoma-in-a-murine-model

Research

JoVE Journal

Cancer Research

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.